Name: high throughput microinjections of sea urchin zygotes
Uploaded: 2014-01-21T21:00:00
Description: Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

Vi takker Santiago Suarez for kritisk læsning af manuskriptet og Betty Cowgill om støtte i fotografering. Vi takker også de anonyme anmeldere for deres kritisk feedback. Dette arbejde understøttes af University of Delaware Research Fund.

1.. Udarbejdelse af protaminsulfat (PS) Coated Dishes <ol><li> Klargør 1% opløsning af protaminsulfat (PS) ved tilsætning af 0,5 g PS i 50 ml deioniseret, destilleret vand (ddH2O) i en 50 ml konisk rør. Ryst godt ved høj hastighed på en bænk ryster ved stuetemperatur i 1-2 timer for at sikre fuldstændig opløsning af PS. Denne løsning kan opbevares ved 4 ° C i mindst 3 måneder (sørg for at opløse gel-lignende bundfald før hver brug) 3.. </li><li> Tag et ærme på 60 mm x 15 mm polys...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offerings+from+an+Urchin.">Offerings from an Urchin.</a> Dev. Biol. 358, 285-294 (2011).</li><li>McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+sea+urchins.">Evolutionary crossroads in developmental biology: sea urchins.</a> Development. 138, 2639-2648 (2011).</li><li>Sodergren, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+article+-+The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">Research article - The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Roux, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B+synthesis+is+required+for+sea+urchin+oocyte+maturation.">Cyclin B synthesis is required for sea urchin oocyte maturation.</a> Dev. Biol. 256, 258-275 (2003).</li><li>Wong, J. L., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+modifications+at+fertilization:+regulation+of+dityrosine+crosslinking+by+transamidation.">Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation.</a> Development. 136, 1835-1847 (2009).</li><li>Shuster, C. B., Burgess, D. 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Mikroinjektion er en kraftfuld teknik til at levere forskellige behandlinger såsom DNA, mRNA, rekombinante proteiner, tab af funktion og forstærkning funktions reagenser, farvestoffer og kombinationer i æg, embryoner, og celler af forskellige organismer 1-7. Dog bør flere overvejelser skal holdes i tankerne, når designe en mikroinjektion eksperiment. Det er kritisk vigtigt at overveje opløseligheden af ​​den leverede behandling og injektionsvolumen. Hvis mikroinjiceret l...

Effektiv og reproducerbar behandling levering er en af ​​de vigtigste metodiske udfordringer for forskere. Der er etableret flere metoder til forbigående levere behandling løsninger ind i de æg, embryoner og celler. Disse metoder omfatter elektroporation (baseret på en genererer forbigående porer i membranen ved hjælp af korte elektriske impulser) 1,2, lipofection (levering ved fusion af behandlings-holdige liposomer med membranen) 1, mikropartikelbombardement 1 (DNA udfældes på micron mellemstore metalpartikler, der derefter bruges til at trænge ind i cellerne ved høj hastighed) og transduktion (virus anvendes som fremføringsmiddel af transgener). I øjeblikket, mikroinjektion er den eneste metode, der besidder den fordel, at der afgives en opløsning med 100% effektivitet med minimale reagenser. Desuden kan en enkelt injektion opløsning består af en kompleks blanding af behandlinger. Denne teknik har været anvendt til vellykketly microinject æg og embryoner fra talrige arter såsom søpindsvin 3,4, zebra fisk 5, mus 6, frø 7 og kvæg 8, samt enkelte celler i vævskultur 9. Enlige blastomeres injektioner på senere udviklingsstadier er også blevet gennemført 10-12. De nuværende metoder til microinjections er baseret på tryk-injektion metode, der oprindeligt blev beskrevet af Hiramoto 10, men er blevet gjort store fremskridt i retning af en optimering af denne proces. Fremragende Mikroinjektionsteknikker er blevet beskrevet andetsteds 11, og her beskriver vi en af de konkrete metoder, der i øjeblikket anvendes til microinject søpindsvin (Strongylocentrotus purpuratus) nyligt befrugtede æg. I over et århundrede har søpindsvin været en værdifuld eksperimentel model 15,16. Søpindsvin er evolutionært tæt knyttet til chordater (inklusive os) og analyse afderes genom afslørede, at de indeholder alle de store gen familier som den menneskelige 17. De producerer et stort antal synkront udvikling af gennemsigtige embryoner, der let kan manipuleres. Brug Søpindsvin som en model organisme har søpindsvin samfund bidraget til vores forståelse af befrugtning proces 18-21, cellebiologiske processer 22-24, og de ​​regulerende netværk gen (GRNs) 25-28. Mikroinjektion i søpindsvin zygoter kræver flere trin. Først skal immobiliseres før injektioner (beskrevet nedenfor) æggene. Mikroinjektion retter overtrækkes med protaminsulfat (PS), som skaber et positivt ladet overflade, hvortil negativt ladede æg kan klæbe 3. Æggene dejellied ved inkubering i surt havvand (pH 5,15) i 10 minutter, efterfulgt af to vaske i naturligt havvand eller kunstigt havvand (pH 8,0). De dejellied æg er omhyggeligt roet i en lige linjei midten af PS-belagt skål i nærvær af 1 mM 3-aminotriazol (3-AT), som er nødvendig for at hæmme aktiviteten af ovoperoxidase der udskilles fra corticale granuler af ægget som et resultat af befrugtning 29. Dette trin er vigtigt for at forhindre hærdning af befrugtningen kuvert og lette mikroinjektion nål post. Som et alternativ til 1 mM 3-AT, kan 10 mM paraminobenzoic syre (PABA) anvendes. Injektionsvæsken er indlæst i en mikroinjektion nål ved hjælp af specialiserede microloading pipettespids og monteret på en holder fastgjort til mikromanipulator og trykenhed (figur 1). Hver nål kan anvendes til at microinject individuelle zygoter på flere forsøg på separate dage. Mikroinjektion kan udføres i 10-15 minutter, indtil zygoter hærde. De zygoter vaskes derefter med havvand og dyrket ved 15 ° C. Når embryonerne nå udrugning blastulaen tidspunkt de frigiver udrugning enzym, der fordøjer komponenter af fertilization kuvert 30 og give dem mulighed for at naturligt løsne sig fra PS-belagt fad. Hvis det er nødvendigt, kan embryonerne forsigtigt løsrevet fra skålen ved hjælp af en mund pipette eller Pasteur-pipette ved forsigtigt at blæse havvand på embryoner. Den beskrevne metode muliggør effektiv og pålidelig mikroinjektion af 100-400 nyligt befrugtede æg på et enkelt fad, hvilket giver en high-throughput metode til downstream analyser.

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

high throughput microinjections of sea urchin zygotes

Mikroinjektion i celler og embryoer er en almindelig teknik, der anvendes til at studere en bred vifte af biologiske processer. I denne fremgangsmåde en lille mængde af behandlingsopløsningen er indlæst i en mikroinjektion kanyle, der anvendes til fysisk at injicere individuelle immobiliserede celler eller embryoner. Trods behovet for indledende uddannelse til at udføre denne procedure for high-throughput levering, mikroinjektion giver maksimal effektivitet og reproducerbar afgivelse af en bred vifte af behandling løsninger (herunder komplekse blandinger af prøver) i celler, æg eller embryoner. Ansøgninger til microinjections omfatter levering af DNA-konstruktioner mRNAs, rekombinante proteiner, gevinst på funktion og tab af funktion reagenser. Fluorescerende eller kolorimetrisk farvestof tilsættes til den indsprøjtede opløsning at muliggøre øjeblikkelig visualisering af effektiv levering samt et redskab til pålidelig normalisering af mængden af ​​den leverede løsning. Den beskrevne metode giver mulighed for mikroinjektion af 100-400 Søpindsvin zygotes inden for 10-15 min.

GFP og mCherry reporterkonstruktioner blev in vitro-transkriberet og mikroinjiceret i de nyligt befrugtede æg. Embryoer blev inkuberet ved 15 ° C i 24 timer (indtil blastulaen etape) og afbildet ved hjælp af Zeiss Observer Z1 mikroskop. Injektion af reporterkonstruktioner førte ikke til nogen udviklingsmæssige defekter (figur 6). Til kvantificering af fluorescerende signaler blev billede erhvervelse udføres ved lav forstørrelse (100X) til maksimalt optage fluorescerende pixel (fi...

Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

High Throughput Microinjections of Sea Urchin Zygotes

Mikroinjektion er en almindelig teknik, der anvendes til at levere DNA-konstruktioner, mRNA&#39;er, morpholino antisense-oligonucleotider eller andre behandlinger i æg, embryoner, og celler fra forskellige arter.

High Throughput Microinjections af søpindsvin Zygoter

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

high-throughput-microinjections-of-sea-urchin-zygotes

Research

JoVE Journal

Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Oprensning af Affinity-mærkede rekombinante proteiner

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Flowcytometri-baseret oprensning af<em&gt; S. cerevisiae</em&gt; Zygoter

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

High-throughput Titrering af luciferase-udtrykkende rekombinante vira

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Metode til Mikroinjektion af<em&gt; Patiria minata</em&gt; Zygoter

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Inddrivelse af voksne zebrafisk Hearts for high-throughput applikationer

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

High-throughput screening for kemiske modulatorer af post-transkriptionelt regulerede gener

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identifikation af kinase-substrat Pairs Brug højkapacitetsscreening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...